This invention relates to tetrazole derivatives which have utility as ligands for opioid receptors.
More particularly, this invention relates to tetrazoles, their preparation and their use as selective agonists for the delta receptor.
At least three subtypes of opioid receptors (mu, delta and kappa) are described and documented in the scientific literature. All three receptors are to be present in the central and peripheral nervous systems of many species including man. Activation of delta receptors is known to produce antinociception in rodents and can induce analgesia in man, in addition to influencing motility of the gastrointestinal tract [see Burks, T. F. (1995) in xe2x80x9cThe pharmacology of opioid peptidesxe2x80x9d, Tseng L. F. ed. Harwood Academic Publishers].
We have discovered a novel class of tetrazole derivatives which are potent and selective delta opioid agonists which are useful for preventing or treating inflammatory diseases such as arthritis, psoriasis, asthma, or inflammatory bowel disease, disorders of respiratory function, gastrointestinal disorders such as functional bowel disease, functional GI disorders such as irritable bowel syndrome, functional diarrhoea, functional distension, functional pain, non-ulcerogenic dyspepsia or others associated with disorders of motility or secretion, urogenital tract disorders such as incontinence, as analgesics for treating pain including non-somatic pain, or as immunosuppressants to prevent rejection in organ transplant and skin graft. Thus the invention provides compounds of the formula: 
and their pharmaceutically acceptable salts; wherein
R1 is H, C2-C6 alkanoyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, (C3-C7 cycloalkyl)-(C1-C4 alkyl), (C1-C4 alkoxy)-(C1-C4 alkyl), carboxy-(C1-C4 alkyl), aryl-(C1-C4 alkyl) or heteroaryl-(C1-C4 alkyl);
R2 and R3 are each independently H or C1-C4 alkyl;
R4 is selected from (i) H, (ii) a group of the formula R6xe2x80x94(CH2)mxe2x80x94Zxe2x80x94(CH2)nxe2x80x94, where m is 0, 1, 2 or 3, n is 1, 2 or 3, Z is a direct link or O, and R6 is xe2x80x94CO2H or xe2x80x94CO2(C1-C4 alkyl), and (iii) a group of the formula 
xe2x80x83where R7 is H or C1-C4 alkyl;
and R5 is hydroxy, C1-C4 alkoxy or xe2x80x94NHSO2(C1-C4 alkyl);
with the proviso that when Z is O, m is 1, 2 or 3 and n is 2 or 3.
Where appropriate, the alkyl alkanoyl, alkoxy, alkenyl and alkynyl groups can be straight or branched chain.
Preferred aryl groups are phenyl and naphthyl, both optionally substituted by up to three substituents each independently selected from halo, trifluoromethyl, C1-C4 alkyl and C1-C4 alkoxy.
More preferably, xe2x80x9carylxe2x80x9d is phenyl optionally substituted by one or two substituents as defined above.
xe2x80x9cHaloxe2x80x9d means F, Cl, Br or I.
Preferred heteroaryl groups include 5- or 6-membered aromatic heterocyclic groups such as thiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl and pyrazolyl.
Thiazol-2-yl is the most preferred heteroaryl group.
The preferred alkyl groups are methyl and ethyl. The preferred alkoxy groups are methoxy and ethoxy The preferred alkanoyl group is acetyl. The preferred alkenyl group is allyl. The preferred alkynyl group is vinyl. The preferred cycloalkyl group is cyclopropyl.
The tetrazole group is preferably attached to the 3- or 4- position of the adjacent phenyl ring.
R1 is preferably H, allyl, benzyl, C1-C4 alkyl, or (C3-C7 cycloalkyl)methyl; most preferably allyl.
R2 and R3 are preferably each independently H or methyl; more preferably both methyl or both H; most preferably both methyl.
R5 is preferably hydroxy, methoxy or xe2x80x94NHSO2Me; most preferably hydroxy.
R4 is preferably H or a group of the formula (a) xe2x80x94(CH2)pCO2H or xe2x80x94(CH2)pCO2 (C1-C4 alkyl) where p is 1, 2, 3 or 4, (b) xe2x80x94(CH2)2xe2x80x94Oxe2x80x94CH2CO2H, (c) xe2x80x94(CH2)2xe2x80x94Oxe2x80x94CH2CO2(C1-C4 alkyl) or (d), 
where R7 is H or C1-C4 alkyl.
In R4, the preferred alkyl group is ethyl.
Preferred individual compounds are those of Examples 1, 4, 24, 27, 36, 42, 94, 96, 104 and 107.
The preferred stereo chemistry of the compounds of the formula (i) is as follows: 
Such compounds are most readily prepared by using starting materials with the appropriate stereochemistry.
The compounds of the formula (I) can be prepared by conventional routes such as those set out in the following Examples and Preparations and in WO-A-9315062.
Route A
Compounds of the formula (I) in which R5 is hydroxy can be prepared by the reaction of the corresponding methoxy compounds of the formula (I) with boron tribromide. Preferably boron tribromide in dichloromethane is added to a solution of the methoxy starting material in dichloromethane and the mixture is stirred at room temperature for a few hours. The product can then be isolated and purified by conventional techniques.
Removal of a hydroxy-protecting group from the corresponding hydroxy-protected compound is also possible, typified by the conversion of t-butyldimethylsilyloxy to hydroxy using tetraethylammnonium fluoride.
Route B
The compounds (I) in which R4 is H, can be prepared by the reaction of a corresponding nitrile of the formula: 
where R1, R2, R3 and R5 are as defined for formula (I), with dibutyltin oxide and trimethylsilyl azide.
The reaction is typically carried out in a suitable organic solvent such as dry toluene at from about 50xc2x0 C. to the reflux temperature. If necessary, a hydroxy group represented by R5 can be protected prior to reaction, e.g. by a t-butyldimethylsilyl protecting group, and the protecting group can be removed subsequently by a conventional technique.
Route C
Compounds (I) in which R4 is either (i) R6xe2x80x94(CH2)mxe2x80x94Zxe2x80x94(CH2)nxe2x80x94 where R6 is xe2x80x94CO2(C1-C4 alkyl) and Z, m and n are as defined for formula (I) or (ii) 
where R7 is C1-C4 alkyl can be prepared by the alkylation of the corresponding compounds in which R4 is H with an alkylating agent of the formula, respectively,
R6xe2x80x94(CH2)mxe2x80x94Zxe2x80x94(CH2)nxe2x80x94Q1xe2x80x83xe2x80x83(IIIA)
or 
where Z, m and n are as defined for formula (I), R6 and R7 are defined in this method and Q1 is a leaving group, preferably Br.
The reaction is typically carried out in the presence of a base such as potassium or cesium carbonate in a suitable organic solvent, e.g. acetonitrile, under gentle reflux.
This reaction generally produces a mixture of compounds in which the group R4 is attached to the 1- and 2- positions of the tetrazole ring.
When a compound in which R5 is hydroxy is required, it may be necessary to protect the hydroxy group before reaction, such as by a t-butyldimethylsilyl group, which can be removed conventionally after reaction, e.g. by the use of tetraethylammonium fluoride.
In an alternative alkylation procedure, the corresponding compound in which R4 is H is reacted firstly with a strong base such as sodium ethoxide (prepared by adding sodium metal to ethanol) and then with the compound III, no additional base being necessary.
Route D
Compounds (I) in which R6 is xe2x80x94CO2H can also be prepared by the hydrolysis, preferably alkaline hydrolysis, of the corresponding esters in which R6 is C1-C4 alkyl.
The reaction is typically carried out with aqueous sodium hydroxide in methanol or a mixture of dioxane and methanol at room temperature.
Route E
Compounds (I) in which R6 is xe2x80x94CO2H can also be prepared by the hydrolysis of the corresponding compounds in which R4 is NCxe2x80x94(CH2)mxe2x80x94Zxe2x80x94(CH2)nxe2x80x94 where Z, m and n are as defined for formula (I). Acidic hydrolysis using hydrogen chloride gas in ethanol is preferred.
Route F
Compounds (I) in which R4 is 1-[CH2CO2(C1-C4 alkyl)] and R5 is xe2x80x94OH can be prepared by ring closure by the reaction of a compound of the formula: 
where R1, R2 and R3 are as defined for formula (I) and Q2 is a hydroxy-protecting group such as t-butyldimethylsilyl, with diethyl acidodicarboxylate, triphenyl phosphine and trimethylsilyl azide in a suitable organic solvent such as toluene. Generally the protecting group Q2 is removed under the reaction conditions.
Route G
Compounds (I) can be prepared by reaction of an aldehyde of the formula: 
where R4 is as defined for formula (I), with a compound of the formula: 
where R1, R2 and R3 are as defined for formula (I), in the presence of benzotriazole, typically under reflux in an organic solvent such as toluene with azeotropic removal of water, following by cooling, e.g. to xe2x88x9220xc2x0 C., and reaction with a Grignard reagent of the formula: 
where R5 is xe2x80x94OQ2 where Q2 is a hydroxy-protecting group such as trimethylsilyl.
Any hydroxy-protecting groups which are present are generally removed by the reaction conditions, or can be subsequently removed by a conventional technique.
Route H
Compounds in which R1 is H can be prepared by reaction of the corresponding compound in which R1 is allyl with tris(triphenylphosphine)rhodium(I) chloride, typically under gentle reflux in a solvent system such as aqueous acetonitrile.
Route I
Compounds of the formula (I) in which R4 is R6xe2x80x94(CH2)mxe2x80x94Zxe2x80x94(CH2)nxe2x80x94 attached to the 1-position of the tetrazole ring and Z,m,n,R1,R2,R3,R5 and R6 are as defined for formula (I) can be prepared as follows: 
Route J
Compounds of the formula (I) in which R5 is xe2x80x94NHSO2(C1-C4 alkyl) can be prepared by the reaction of the corresponding amino-substituted compound with a C1-C4 alkanesulphonyl chloride, typically in the presence of an acid-acceptor.
Route K
Compounds of the formula (I) in which R1 is C2-C6 alkyl, aryl-C1-C4 alkyl) or heteroaryl-(C1-C4 alkyl) can be prepared by the reductive alkylation of the corresponding compounds in which R1 is H using the appropriate aldehyde (C1-C5 alkyl)CHO, aryl CHO or heteroaryl CHO and a reducing agent such as sodium triacetoxyborohydride.
The invention also includes any novel intermediates described herein, particularly those of the formula (II).
The necessary intermediates for the processes described above can be prepared by conventional methods such as those set out in the following Preparations, e.g. as follows: 
R1, R2 and R3 are as defined for formula (I); TBS=t-butyldimethylsilyl. 
R1, R2 and R3 are as defined for formula (I) and R5 is C1-C4 alkoxy or xe2x80x94NHSO2(C1-C4 alkyl). 
Suitable pharmaceutically acceptable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, hydrogen sulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, benzoate, methanesulphonate, benzenesulphonate and p-toluenesulphonate salts.
Suitable pharmaceutically acceptable base salts are formed from bases which form non-toxic salts and examples are the calcium, lithium, magnesium, potassium, sodium, zinc, ethanolarnine, diethanolamine and triethanolamine salts.
For a review on suitable salts see Berge et al, J.Pharm.Sci., 66 1-19 (1977).
As will already be apparent the compounds of the formula (I) will contain one or more asymmetric carbon atoms and will therefore exist in two or more stereoisomeric forms, or they may exist as tautomers. The present invention includes the individual stereoisomers and tautomers of the compounds of the formula (I) and mixtures thereof.
Separation of diastereoisomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of the formula (I) or a suitable salt or derivative thereof An individual enantiomer of a compound of the formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base.
Receptor Binding Assays
Opioid (mu and kappa) receptor binding assays were performed in guinea-pig brain membrane preparations. Binding assays were carried out at 25xc2x0 C. for 60 minutes in 50 mM Tris (pH 7.4) buffer. [3H]-DAMGO (2 nM) and [3H]-U-69,593 (2 nM) were used to label mu and kappa receptor binding sites respectively. The protein concentration was approximately 200 xcexcg/well. Non-specific binding was defined with 10 xcexcM naloxone.
Delta receptor binding assay was performed in a stable line of CHO cells expressing the human delta receptor. The binding assay was carried out at 25xc2x0 C. for 120 minutes in 50 mM Tris (pH 7.4) buffer. [3H]-SNC-80 was used to label delta receptor binding sites. The protein concentration was approximately 12.5 xcexcg/well. Non-specific binding was defined with 10 xcexcM naltrexone.
The binding reaction was terminated by rapid filtration through glass fiber filters, and the samples washed with ice-cold 50 mM Tris buffer (pH 7.4). All assays were performed in duplicate/triplicate.
Isolated Tissue Studies
Opioid (delta, mu and kappa) activity was studied in two isolated tissues, the mouse vas deferens (MVD)(xcex4) and the guinea-pig myentric plexus with attached longitudinal muscle (GPMP)(xcexc and xcexa).
MVD (DC1 strain, Charles River, 25-35 g) were suspended in 15 ml organ baths containing Mg++-free Krebs"" buffer of the following composition (mM): NaCl, 119; KCl, 4.7; NaHCO3, 25; KH2PO4, 1.2; CaCl2, 2,5 and glucose, 11. The buffer was gassed with 95% O2 and 5% CO2. The tissues were suspended between platinum electrodes, attached to an isometric transducer with 500 mg tension and stimulated with 0.03 Hz pulses of 1-msec pulse-width at supramaxinal voltage. IC50 values were determined by the regression analysis of concentration-response curves for inhibition of electrically-induced contractions in the presence of 300 nM of the mu-selective antagonist CTOP. This test is a measure of xcex4 agonism.
Guinea-pig (Porcellus strain, male, 450-500 g, Dunkin Hartley) myentric plexus with attached longitudinal muscle segments were suspended with 1 g of tension in Krebs"" buffer and stimulated with 0.1 Hz pulses of 1-msec pulse-width at supramaximal voltage. Mu functional activity was determined in the presence of 10 nM nor-BNI with 1 xcexcM of the mu selective agonist, DAMGO, added to the bath at the end of the experiment to define a maximal response. This test is a measure of mu functional agonism.
Kappa functional activity was determined in the presence of and 1 xcexcM CTOP with 1 xcexcM of the kappa selective agonist U-69,593 added at the end of the experiment to define a maximal response. All inhibitions of twitch height for test compounds were expressed as a percentage of the inhibition obtained with the standard agonist and the corresponding IC50 value determined.
DAMGO is [D-Ala2,N-MePhe4,Gly5-ol]enkephalin)
U69593 is ((5a, 7a, 8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)-benzeneacetamide)
SNC-80 is (+)-4-[(xcex1R)-xcex1((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide
norBNI is nor-binaltorphimine
CTOP is 1,2-Dithia-5,8,11,14,17-pentaazacycloeicosane, cyclic peptide derivative
DPDPE is [D-Pen2,D-Pen5]enkephalin)
[3H]-DAMGO, [3H]-U69593, norBNI, and CTOP are all commercially available from DuPont, Amersham International, RBI and DuPont respectively. [3H]-SNC80 was prepared by Amersham International.
In general, a therapeutically effective daily oral or intravenous dose of the compounds of formula (I) and their salts is likely to range from 0.01 to 50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg. The compounds of the formula (I) and their salts may also be administered by intravenous infusion, at a dose which is likely to range from 0.001-10 mg/kg/hr.
Tablets or capsules of the compounds may be administered singly or two or more at a time, as appropriate. It is also possible to administer the compounds in sustained release formulations.
The physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Alternatively, the compounds of the formula (I) can be administered by inhalation or in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. An alternative means of transdermal administration is by use of a skin patch. For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.